This is an application for continuation of a long-term project to comprehensively identify and functionally analyze protein interactions and posttranslational modifications regulating to eukaryotic protein synthesis. Protein synthesis is a fundamental process essential for replication, growth, and maintenance of all living organisms. Aberrant protein synthesis contributes to a number of human diseases including cancer and mental retardation. Hence a comprehensive understanding of protein synthesis is needed for systems analysis of both the normal and disease-altered translation process. Our strategy combines tandem mass spectrometry-based proteomics with genetic and biochemical functional assays to identify and dissect the function of uncharacterized proteins and posttranslational modifications controlling translation. Initially, we identified and validated a large number of uncharacterized proteins and posttranslational modifications associated with ribosomes, polysomes, and translation factors in S. cerevisiae. In this application, experiments are proposed to determine the function of a subset of these proteins linked to translation initiation. Initiation is the main step in which translation is regulated. First, we will assay yeast deletion mutants using sensitive biochemical and genetic experiments to identify defects in translation initiation. To complement the genetic and biochemical assays, we will perform synthetic genetic analysis on a genome- wide scale to genetically identify interacting proteins. By combining these approaches, we expect to develop functional models for many of uncharacterized proteins. Second, we will identify selected posttranslational modifications on translation initiation factors. Mass spectrometry will be used to specifically identify the modified amino acids. These amino acids will be mutated to establish their biological significance. Finally, we will extend our interest in identifying translation initiation factors to the poorly understood process of cap- independent initiation. The essential cell-cycle gene ornithine decarboxylase (ODC) and the oncogene c- myc are translated through a cap-independent mechanism using internal ribosomal entry sites (IRES). We will use novel proteomic screens to identify proteins that bind specifically to these mRNA sites. Reporter assays will be used be to test candidate proteins for positive and negative effects on ODC and c-myc cap- independent translation.